There is a growing family of proteins which share regions of sequence homology and localization to the nucleus. These proteins include the thymopoietins, (Zevin-Sonkin et al., Immuno. Letts. 31:301-10, 1992; Harris et al., Proc. Natl. Acad. Sci. USA 91:6283-87, 1994; Harris et al., Genomics 28:198-205, 1995; Berger et al., Genome Res. 6:361-70, 1996 and Ishijima et al., Biochem. Biophys. Res. Comm. 226:431-8, 1996), lamina associated proteins, (Senior and Gerace, J. Cell Biol. 107:2029-36, 1988; Worman et al., J. Cell Biol. 111:1535-42, 1990; Wozniak and Blobel J. Cell Biol. 119:1441-9, 1992; Foisner and Gerace, Cell 73:1267-79, 1993; Ye and Worman, J. Biol. Chem. 269:11306-11, 1994 and Furukawa et al., EMBO J. 14:1626-36, 1995) and emerin (Bione et al., Nat. Genet. 8:323-7, 1994; Manilal et al., Hum. Mol. Gen. 5:801-8, 1996 and Small et al., Mamm. Genom. 8:337-41, 1997).
Emerin is a nuclear membrane protein responsible for the X-linked recessive disorder Emery-Dreifuss muscular dystrophy. Mouse, rat and human emerin sequences have been reported (Bione et al., Nat. Genet. 8:323-7, 1994; Manila et al., Hum. Mol. Genet. 5:801-8, 1996 and Small et al., Mammal. Genom. 8:337-41, 1997). The mouse, rat and human emerin share 73-95% nucleotide and amino acid identity. All share some structural homology with the thymopoietins and LAP2, in particular within portions of the conserved N-terminal region and the hydrophobic putative transmembrane domain of thymopoietin. Like the thymopoietins and LAP2, emerin is ubiquitous expressed and it is predicted that emerin has the same inner nuclear membrane organization as do thymopoietin and LAP2 (Manilal et al., ibid.). Antisera raised against emerin peptides localized expression of the protein to the nuclear membranes of normal skeletal and cardiac muscle cells, but found it to be absent in those cells of patients with muscular dystrophy. It is unclear how a deficiency of a nuclear protein results in the disease (Nagano et al., Nat. Genet. 12:254-9, 1996 and Small et al., ibid.).
The present invention provides associated polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 1 through 876 of SEQ ID NO:2. Within one embodiment the sequence of amino acid residues is at least 90% identical. Within another embodiment any differences between said polypeptide and residues 1 through 876 of SEQ ID NO:2 are due to conservative amino acid substitutions. Within another embodiment the polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2. Within a further embodiment the polypeptide is covalently linked to a moiety selected from the group consisting of affinity tags, radionucleotides, enzymes and fluorophores. Within a related embodiment the moiety is an affinity tag selected from the group consisting of polyhistidine, FLAG, Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region.
Also provided is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2.
Within another aspect the invention provides a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion consisting of a polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 1 through 876 of SEQ ID NO:2; and said second portion comprising another polypeptide.
Within yet another aspect the invention provides a pharmaceutical composition comprising a polypeptide as described above, in combination with a pharmaceutically acceptable vehicle.
Within still another aspect is provided an antibody or antibody fragment that specifically binds to a polypeptide as described above. Within one embodiment the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b); and d) human monoclonal antibody. Within another embodiment the antibody fragment is selected from the group consisting of F(abxe2x80x2), F(ab), Fabxe2x80x2, Fab, Fv, scFv, and minimal recognition unit. Within still another embodiment is provided an anti-idiotype antibody that specifically binds to the antibody described above.
Also provided is a binding protein that specifically binds to an epitope of a polypeptide as described above.
Within another aspect of the invention is provided an isolated polynucleotide selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 1 through 876 of SEQ ID NO:2; b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5; c) a polynucleotide that remains hybridized following stringent wash conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1, or the complement of SEQ ID NO:1. Within one embodiment the sequence of amino acid residues is at least 90% identical. Within another embodiment any difference between the amino acid sequence encoded by the polynucleotide and the corresponding amino acid sequence of SEQ ID NO:2 is due to a conservative amino acid substitution. Within yet another embodiment the polynucleotide comprises nucleotide 127 to nucleotide 2754 of SEQ ID NO:1. Within still another embodiment the polynucleotide is DNA.
Within another aspect the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment consisting of a polynucleotide as described above; and a transcriptional terminator. Within one embodiment the sequence of amino acid residues is at least 90% identical. Within another embodiment any difference between the amino acid sequence encoded by the polynucleotide and the corresponding amino acid sequence of SEQ ID NO:2 is due to a conservative amino acid substitution. Within another embodiment the DNA segment encodes a polypeptide covalently linked to an affinity tag selected from the group consisting of polyhistidine, Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region. Within yet another embodiment the expression vector further comprises a secretory signal sequence operably linked to said DNA segment.
Also provided is a cultured cell into which has been introduced an expression vector as described above, wherein the cell expresses the polypeptide encoded by the DNA segment.
Within a further aspect the invention provide a method of producing a ZTMPO-1 polypeptide comprising: culturing a cell into which has been introduced an expression vector as described above, whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.
Also provided by the invention is a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:1 or the complement of SEQ ID NO:1, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; comparing said first reaction product to a control reaction product, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and attached drawing.